The determination of the intermediate and end products of protein metabolism is important in clinical chemistry, and particularly in the diagnosis of kidney function. The products of this metabolism include creatinine and creatine.
Creatinine is a product of the endogenous metabolism of muscle. The amount of creatinine in the urine reflects total muscle mass and the degree of muscle activity. The amount of creatinine in a person's urine is generally constant, varying little from day to day.
Urinary creatine is elevated in the early stages of muscular dystrophy, when muscle destruction is occurring rapidly, and in any wasting disease involving increased tissue catabolism. It is elevated during severe and strenuous muscular activity and in hyperthyroidism. Urinary creatinine is decreased during the later stages of muscular dystrophy and whenever renal function is impaired. Urinary creatine increases in the same disease states that produce an increase in urinary creatinine.
Methods for the determination of creatinine have been frequently described. Early tests were based on the nonenzymatic Jaffe reaction which involves the formation of an orange-red color with an alkaline picrate solution. This method, however, is not specific for creatinine since many creatinine-like substances also react with alkaline picrate.
Enzymatic assays for creatinine have been developed using enzymes specific for creatinine and creatine, respectively, and the following reaction sequence: EQU creatinine+water.revreaction.creatine (1) EQU creatine+water.fwdarw.urea+sarcosine (2)
The first reaction is catalyzed by creatinine amidohydrolase whereas the second one is catalyzed by creatine amidinohydrolase.
An assay for creatinine is described in U.S. Pat. No. 4,215,197 (issued July 29, 1980) wherein the enzymes noted above are used in combination with a tetrazolium indicator. The assay can be performed in solution or with a dry device into which the reagents have been imbibed. Only creatinine can be measured when all of the reagents are included in the analytical composition. As shown in Example I (Col. 5, line 64 to Col. 6, line 24) of this reference, the assay requires a long incubation period (i.e. up to sixty minutes), and a blanking step to subtract out background resulting from the reagents used and endogenous creatine. The sensitivity is allegedly increased by including formaldehyde dehydrogenase, diaphorase and NAD in the composition. This significantly complicates the assay with additional reagents and enzymatic reactions. Creatine or sarcosine can be measured with the assay if the appropriate enzymes are omitted. But this requires separate compositions or test devices for determining each analyte.
Japanese Patent Publication 58(1983)-009699 (published Jan. 20, 1983) describes an apparatus and method for solution assay of either creatinine or creatine. The assay is quite complex, requiring extensive reaction chambers, fluid pumps and multiple signal detecting equipment. Two readings are taken to determine both analytes. However, the assay does not measure the amount of creatinine independently, but rather as the difference between two end point measurements.
It would be desirable to have a relatively simple, automated dry assay that is equally useful for either creatinine or creatine determination, which is highly sensitive and rapid and requires no blanking step.